A long-term goal of the research in our laboratory is to re-create the components and compartments of mammalian systems, and in particular human systems, to engineer functional hybrid models. The initial period for this Bioengineering Research Grant (BRG) was to investigate the motoneuron to muscle segment of the reflex arc to understand in vitro what are possible outcomes when a human stem cell is implanted in a rat model and then apply it to improve the outcomes in an in vivo system. We have made a number of breakthroughs with this model system that move us closer to the goal of creating a functional in vitro model of the reflex arc: 1) We have cultivated embryonic and adult rat motoneurons as well as motoneurons derived from human fetal stem cells (hFSC). 2) We have demonstrated neuromuscular junction formation between these rat and human derived motoneurons with myotubes derived from both rat and human adult satellite cells. However, we have also created mature myotubes that exhibit sarcomere formation, express myosin heavy chain, have established the EC coupling apparatus as well as Type I and Type II muscle isoforms. We have also differentiated the myotubes into intrafusal fibers and integrated then with the MEMS constructs and demonstrated that these are functional mechanoreceptor synapses. 3) Glial cells have been incorporated into our model system and we have demonstrated Schwann cell myelination of motoneurons, with complete Node of Ranvier formation, and derived pure populations of MBP (myelin basic protein) positive oligodendrocytes from human mesenchymal stem cells. 4) We have successfully demonstrated in vivo integration of the differentiated hFSC derived motoneurons into an adult rat spinal cord injury model and shown significant locomotor recovery as accessed via the BBB test scoring system. This renewal BRG application seeks to complete the development of the entire reflex arc in vitro using primary rat cells and then with cells derived from human stem cells. We will also initiate experiments to increase the throughput of first the motoneurons to muscle segment of the stretch reflex are and then for the entire reflex arc. The Hypothesis is that we can recreate a functional in vitro model of the reflex arc with primary rat cells and achieve the same result with cells derived from human stem cells that will be a prototype for the next generation screens for drug discovery and toxicology. The continuation of the grant will enable advances for 1) the creation of a human cell based reflex arc in a defined, serum-free environment and 2) the development of new technology for utilization in the next level of Systems Biology and high-information content screens. Our expertise in surface chemistry, Micro-ElectroMechanical Systems (MEMS) fabrication and developmental/cellular/molecular biology has now allowed the construction of complex functional neuronal systems in vitro. Our team possesses the necessary expertise in all aspects of this project, as well as the facilities to effectively and successfully perform the research. PUBLIC HEALTH RELEVANCE: The goal of this project is to engineer a system to model one of the most fundamental motor circuits in the human body, the spinal reflex arc. We will use nanotechnology and microelectronics in combination with biomedical engineering techniques to build this hybrid biological/non-biological system. Potential benefits include prevention, diagnosis, and treatment of developmental abnormalities in the spinal cord, rehabilitation of chronic neurological/muscle disorders and accompanying pain, and new strategies for prosthetic and orthotic design and evaluation.